Antigen molecules are recognized by the immune system after internal processing by antigen-presenting cells (APCs) (Lanzavecchia, 1996, Curr. Opin. Immunol., 8:348-54). In order to present an antigen, the antigen is broken down into small peptidic fragments by enzymes contained in vesicles in the cytoplasm of the antigen-presenting cells (for reviews, see: Wick, et al., 1999, Immunol. Rev., 172:153-62; Lehner, et al., 1998, Curr. Biol., 8: R605-8; Braciale, 1992, Curr. Opin. Immunol., 4:59-62). The enzymes are part of a complex of proteolytic enzymes called a proteosome. Most cells have several different types of proteosomes with differing combinations of specificities, which they use to recycle their intracellular proteins. The peptides produced by the proteosomes are generated in the cytosol and must be transported into the Golgi compartment in order to associate with newly synthesized class I molecules. This is accomplished by a heterodimeric protein called TAP (for transporter associated with antigen processing) (Townsend, et al., 1993, Eur. J. Immunogenetics, 19:45-55), which is associated with the ER and actively transports peptides into the Golgi, where they are linked to cellular major histocompatibility complex (MHC) molecules (known as HLA in human).
There are two types of MHC molecules used for antigen presentation, class I and class II molecules. MHC class I molecules are expressed on the surface of all cells and MHC class II are expressed on the surface of a specialized class of cells called professional antigen-presenting cells. MHC class II molecules bind primarily to peptides derived from proteins made outside of an antigen-presenting cell, but can present self (endogenous) antigens. In contrast, MHC class I molecules bind to peptides derived from proteins made inside a cell, including proteins expressed by an infectious agent (e.g., such as a virus) in the cell and by a tumor cell. When the MHC class I proteins reach the surface of the cell these molecules will thus display any one of many peptides derived from the cytosolic proteins of that cell, along with normal “self” peptides being synthesized by the cell. Peptides presented in this way are recognized by T-cell receptors which engage T-lymphocytes in an immune response against the antigens (cellular immunity).
Antigen binding also requires the interaction of a number of co-receptor/ligand molecules that interact with ligand/receptors on the T cell. CD4 and CD8 act as co-receptors (one type only present per T cell) that interact with the TCR on the appropriate T cell to form a receptor/co-receptor complex. The receptor/co-receptor complex binds to the relevant MHC molecules on the APC. CD4 binds to class II molecules and CD8 binds to class I molecules. Various adhesion molecules (e.g., LFA-1, LFA2 (CD2), LFA3 (CD58), ICAM1, ICAM2, ICAM3), costimulatory molecules (e.g., CD80: B7-1 and B7-2) and accessory molecules (e.g., CD83) are also involved in facilitating T cell binding to APCs.
Conventional immunization techniques, such as those using killed or attenuated viruses, often fail to elicit an appropriate CTL response which is effective against an intracellular infection. Thus, there remains a need for the development of vaccines that stimulate appropriate responses (i.e., cell-mediated as well as antibody-mediated immune responses), in order to prevent disease.
Induction of primary MHC class I restricted CTL by pure soluble antigenic proteins in vitro has not been reported. The most common tool for ex vivo induction of primary CTL are small (8-11-mer) synthetic peptides (Stauss, et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89:7871-5); Carbone, et al., 1988, J. Exp. Med., 167:1767-79). These synthetic peptides associate with class I molecules on the cell surface without the requirement for endogenous processing. When presented on the surface of an appropriate APC (such as a dendritic cell) they can then induce a primary CTL response. However, frequently these CTL do not protect against challenge with pathogens that endogenously synthesize the protein from which the peptide was derived because of their low T-cell receptor avidity (Speiser, et al., 1992, J. Immunol., 149:972-80) and because they induce reactivity with a single epitope of the target antigen.
Another way of activating an efficient immune response against a specific antigen is to stimulate T cells with APCs engineered to express a specific antigen. U.S. Pat. Nos. 5,962,320, 6,187,307, 6,194,205 and patent publication WO 97/29183 disclose a method of making engineered APCs by transfecting professional or non-professional APCs with selected antigens to regulate the immune response of a subject.
WO 96/27392, U.S. Pat. Nos. 5,225,042, 6,251,627 and 5,962,320 disclose engineered APCs transfected with MHC molecules.